Despite being caused by a single protein, prion diseases are strikingly heterogenous. Individual prion variants, known as strains, possess distinct biochemical properties, form aggregates with characteristic morphologies and preferentially seed certain brain regions, causing markedly different disease phenotypes. Strain diversity is determined by protein structure, post-translational modifications and the presence of extracellular matrix components, with single amino acid substitutions or altered protein glycosylation exerting dramatic effects. Here, we review recent advances in the study of prion strains and discuss how a deeper knowledge of the molecular origins of strain heterogeneity is providing a foundation for the development of anti-prion therapeutics.